In response to a recently published article by Schaade et al. (12) describing the value of LightCycler technology for quantitative analysis of cytomegalovirus (CMV) in clinical material, we wish to add our experience. Using a different LightCycler (LC; Idaho Technology Inc., Idaho Falls, Idaho), we too have developed a real-time quantitative PCR (QPCR) assay for CMV (11). Our assay targets a shorter fragment of the glycoprotein B gene (150 bp) and incorporates a cyanine 5-labeled reverse primer and a sequence-specific fluorescein-labeled hybridization probe, allowing the identification of PCR product by fluorescence resonance energy transfer. We have designed a quantitative standard (plasmid cloned) with a 2-bp mismatch in the probe-binding region to facilitate its differentiation from CMV target sequence by using melting curve analysis (2, 3). The assay is sensitive (detection limit ≤ 10 copies), with an overall dynamic range of 2 × 103 to 5 × 108 copies/ml. When applied to blood samples, the results compared well with an in-house modification of a qualitative assay (5) and the quantitative data correlated (r = 0.88; P < 0.001) with an independent TaqMan-based QPCR assay (9).
PCR has previously proved suitable for the detection and quantification of CMV DNA in urine (4, 7, 10), whereas standard detection of early antigen fluorescent foci (DEAFF) testing (8) is slow (up to 72 h), lacks sensitivity, and may be hampered by cytotoxicity (1). We therefore sought to extend the clinical utility of our assay to the examination of urine and respiratory samples (including sputum, bronchoalveolar lavage fluid, and nasopharyngeal secretion) to aid the identification of patients at risk of developing CMV disease. We have tested such specimens from pediatric and adult transplant patients, including 46 urine samples (assayed directly, without DNA extraction) and 86 respiratory samples (extracted using the QIAamp DNA minikit; Qiagen, West Sussex, United Kingdom).
QPCR proved to be more sensitive than DEAFF testing, giving three- and five-fold increases in the positivity rate in urine and respiratory samples, respectively, with viral loads ranging from <2 × 103 to 1 × 108 copies/ml (Table 1). All samples were readily evaluated by PCR, whereas 21 of 132 samples (16%) were toxic in the DEAFF test, 7 (33.3%) of which were PCR positive. DNA extraction of urine samples did not improve the positivity rate in our hands (unpublished data), suggesting that direct detection of CMV DNA in urine will suffice, as previously reported (4, 7).
TABLE 1.
Comparison of LightCycler real-time PCR and DEAFF testing for detection of CMV in urine and respiratory samples
| Sample type | Real-time PCR result | No. (%) of samples
|
||
|---|---|---|---|---|
| DEAFFa negative | DEAFF positive | DEAFF toxic | ||
| Urine (n = 56) | Negative | 37 (80.4) | 0 | 0 |
| Positive | 2 (4.4) | 3 (6.5)b | 4 (8.7) | |
| Respiratory (n = 86) | Negative | 51 (59.3) | 0 | 14 (16.3) |
| Positive | 14 (16.3) | 4 (4.6)c | 3 (3.5) | |
Detection of early antigen fluorescent foci.
Viral load range, <2 × 103 to 5 × 104 copies/ml (median, 1.2 × 104).
Viral load range, <2 × 103 to 1 × 108 copies/ml (median, 2.7 × 106).
Our data confirm and extend those of Schaade et al. (12). The LC QPCR assay is rapid (<2 h), provides timely results, and is suitable for the detection of CMV DNA in a range of clinical specimens. The application of LC-based QPCR to testing urine, respiratory, and other specimens, in addition to the determination of CMV viral load in blood, may assist in the early identification of patients with a high risk of progression to CMV disease and in determining appropriate parameters for therapeutic intervention. Prospective monitoring, using QPCR technology to determine the rate of increase in viral load, may refine attempts to identify patients at greatest risk of CMV disease (6).
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